This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our proposed research involves x-ray crystallographic analysis of cytochrome c oxidase (CcO) from Rhodobacter sphaeroides (Rs), the terminal complex of the respiratory chain. We recently obtained high resolution crystal structures of the I-II subunit catalytic core of the RsCcO in the oxidized form at 2.0 [unreadable] resolution, as well as the dithionite-reduced form of the enzyme at 2.2 [unreadable] resolution. In the reduced structure, an unusual displacement of heme a3 group was seen, accompanied by opening of the top of a proton input channel (K path). These changes were not seen in the published bovine heart mitochondrial CcO in the reduced form and could be revealing an important aspect of the gating of the proton pump. However, it is critical to know the actual redox state of the enzyme during data collection. In order to investigate the redox states of the different forms of the crystals, we utilized the on-line microspectrophotometer available at BioCARS, 14-BM-C, to observe the spectral characteristics of RsCcO crystals before, during, and after X-ray irradiation. We observed that for the oxidized form of the enzyme, CuA center and heme a became reduced within a couple of minutes of irradiation, and that another spectral peak at 588 nm started to appear. For the reduced form of RsCcO crystals, the crystal remained reduced during the exposure, with a shoulder peak at approximately 635nm formed within a minute of irradiation. In this run, we will be using small, two-subunit crystals of RsCcO in oxidized and reduced states, as well as the cyanide bound form, in order to investigate the nature of the 588nm species. We will also continue our studies on the spectral changes of the K362M mutant crystals, in comparison with those of the wild type enzyme.